The apical plasma membrane of high resistance epithelia, such as the toad's urinary bladder is the putative passive permeability barrier in the vectorial transepithelial transport of water, sodium and small solutes such as urea. This proposal will capitalize upon our recently developed ability to obtain a preparation enriched in vesicles derived from the apical plasma membrane of this high resistance epithelium. I propose studies on the lipids and proteins in this apical plasma membrane preparation. Lipid studies will include determination of both the composition and the biophysical state (i.e., fluidity) of lipids. The protein studies will be directed toward identification of specific transport functions with specific membrane proteins. Such proteins will be identified two ways. First, covalent reagents which interact with membrane proteins to produce selective effects on physiologic function will be presumed to have reacted with a membrane transport protein. The identity of such a protein will be assessed by gradient slab and two dimensional gel electrophoresis plus autoradiography. The effects of hormones - especially aldosterone and antidiuretic hormone - on the concentration of such transport proteins in the apical membrane will be determined. Second, by the use of protein and lipid cross-linking reagents, the spatial relationships between membrane proteins and lipids will be studied under states of varying function induced by physiologic maneuvers and by hormonal and pharmacologic agents. The long range goals include verification of the functionality of the membrane components by reconstitution of the transport function by incorporation of the protein into functionless lipid vesicles.